The proposed studies are anticipated to yield important new information as to the composition of the bacterial biota that comprises the caries-associated plaque biofilm. The long-term benefit of such information should lead researchers to devising both diagnostic and preventative strategies for dental caries based on addressing its etiological agents. This proposal will focus on children with a severe forms of dental caries called Early Childhood Caries (ECC). Using a powerful technique of gradient electrophoresis will be used to separate 16S rDNA markers from an array of bacteria in plaque biofilms. These gels should show differences between the microfloras of ECC and caries-free children. This profiling, in turn, will allow us to identify or approximate those bacteria, some likely to be uncultivable, associated with caries. Another hypothesis to be tested is whether strains of mutans streptococci, or the entire caries-biofilm differ in their ability to cause disease. Subtraction DNA hybridization will be used to discover unique genetic loci present in mutans streptococci or dental plaques of caries-prone children. Further development of subtraction DNA hybridization will lead to our overall objective, i.e., to characterize from whole plaque a constellation of genetic loci within the caries-active biofilm, irrespective of the limitation of first cultivating specific bacteria. This will set the groundwork for subsequent studies in which a set of DNA probes can be compiled and tested, which will be useful for predicting whether a particular child is at risk for caries. Knowing the function of these genetic loci and the bacterial host from which they arise will give important information as to the causation of caries. Moreover, having genetic markers for disease may eliminate the costly and imprecise practice of cultivating bacteria from dental plaque. The research proposed will likely impact on these more serious forms of caries, leading to its eventual prevention.